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Plant Cell, Tissue and Organ Culture Hort 515
Embryo, Meristem, and Root Cultures
1. Embryo Culture – culture of zygotic embryos to recover plants, i.e. germination of embryos that are dormant or must be rescued at very immature stages of development (hybrids of wide crosses)
2. Meristem Culture – excision and culture of the shoot apical meristem to recover disease-free plants
3. Root Culture – autonomously growing roots for production of secondary products
1. Embryo Culture
I. Germination of dormant embryos - typically the result of either chemicals produced in the ovary/ovule, physical/chemical barriers to seed germination or “dormancy programs”
Seed dormancy requirement may be satisfied by hormone or stratification treatments in vitro
Orchid (epiphyte) seeds do not have an endosperm but nutrients can be supplied in a tissue culture medium (e.g. banana pulp).
II. Rescue of immature embryos - these are products of wide crosses that are exhibiting some incompatibility responses that prevent development of a mature embryo, i.e. products of parents in secondary gene pools, example
Pre-fertilized OvuleAntipodals
Egg
Synergids
Polar nuclei
Two male gametes, one fertilizes the egg to make a zygote and the other fuses with the polar nuclei forming the triploid endosperm.
II. Rescue of immature embryos
Embryo abortion based on zygotic incompatibility barriers
Gene pool classification by Harlan and deWit:
Primary - no genetic barriers to recombination
Secondary - pre- and post-zygotic incompatibility barriers, example
Tertiary - chromosomal barriers that restrict homeologous chromosome pairing and recombination
Incompatibility Barriers
6.
5.
7.
8.
9.
4.
II. Rescue of immature embryos
Embryo abortion based on zygotic incompatibility barriers
Gene pool classification by Harlan and deWit:
Primary - no genetic barriers to recombination
Secondary - pre- and post-zygotic incompatibility barriers,
Tertiary - chromosomal barriers that restrict homeologous chromosome pairing and recombination
II. Rescue of immature embryos
Rescue of immature embryos that are products of wide crosses is possible if the genotypes are members of the secondary gene pool,
i.e. pre- and post-zygotic incompatibility barriers
Test tube fertilization – may result in completion of germination if there are pre-zygotic barriers such as stylar and pollen tube length disparities
Embryo culture – embryo development, germination and seedling development if there are post-zygotic barriers, example
Incompatibility Barriers
6.
5.
7.
8.
9.
4.
4, 5, 6 - may be overcome by test tube fertilization
7, 8, 9 - may be rescued by embryo culture
Embryogenesis - embryo initiation from the zygote; first divisions are horizontal (periclinal), separating the suspensor from the embryo proper and then transverse (anticlinal) divisions begin the process of differentiation, suspensor, proembryo
Embryogeny - embryo development after differentiation, examples
Embryo abortion in wide crosses often occurs during embryogeny (e.g. endosperm degradation) and it is sometimes possible to rescue these embryos and culture in vitro to recover plants
Embryo culture may include the culture of embryos within the ovule or ovary in which instances test-tube fertilization may overcome stigmata or style, and pollen incompatibility barriers
Embryogenesis
Embryogenesis Embryogeny
Embryogenesis - embryo initiation from the zygote; first divisions are horizontal, separating the suspensor from the embryo proper and then transverse divisions begin the process of differentiation, suspensor, proembryo
Embryogeny - embryo development after differentiation
Embryo abortion in wide crosses often occurs during embryogeny (e.g. endosperm degradation) and it is sometimes possible to culture these embryo and recover hybrid plants
Embryo culture may include the culture of embryos within an ovule or ovary in which instances test-tube fertilization may overcome stigmatal or stylar, and pollen incompatibility barriers, examples
Tomato ovary culture
CA poppy ovule culture
Isolation and culture of immature embryos
History - Hannig (1904), 1st embyro culture, Raphanus and Cochlearia on medium containing salts + sucrose
Retention of the ovary on the parent plant
Embryos become more become more autotrophic during development
Plant treatments that facilitate parthenocarpy enhance embryo development, typically facilitated by hormones, example
Isolation and culture of immature embryos
Nutrient Medium
Mineral nutrients – essential micro- and micro-nutrients
Carbohydrates - (carbon source)/osmotic agents, 50 g/L equivalent of sucrose (normal is 20 to 30 g/L), high osmolarity favors embryogeny and prevents premature germination
Growth regulators - Auxin, cytokinin and gibberellins tend to be required for preheart-shape stage embryos
ABA is used to prevent precocious germination, examples
Embryo Culture of Japanese Holly
Embryo Culture of Citrus
2. Meristem Culture for Disease Eradication
Clonal propagation of plants using explants that are free of disease organisms
Typically, the explant is the shoot apex, containing the apical meristem, as this explant often does not contain microbes or viruses and will regenerate shoots; potatoes, strawberries, most tuber crops, citrus
I. Background
Shoot Apical Meristem - apical portion of the shoot that contains the progenitors of vegetative cells and subsequently germ cells
Tunica - peripheral 1 to 3 layers of cells characterized by anticlinal divisions, gives rise to the epidermis/subepidermis
Corpus - cells subjacent to the tunica, periclinal and anticlinal divisions and gives rise to the cortex, vascular system and pith
Meristem initials - 3 to 5 cells that are progenitors of the tunica/corpus, relatively low cell division frequency
Dicot Shoot Apical Meristem
Shoot apex - meristem with leaf initials, most typically is the explant that is cultured for disease eradication, larger in size and more autotrophic than the true apical meristem, example
150 m
Asparagus Shoot Apex
Shoot apical meristem is often free of viruses and other pathogens
Vasculature is not directly connected to the meristem
II. Factors affecting recovery of disease-free plants
Treatment of the donor plant - treatments that favor differential growth of the plant over the disease organism
Gibberellin or etiolation treatments – facilitate more rapid growth of the shoot
Thermotherapy treatment of plants – reduces pathogen growth (viral replication), 35 to 42 C constant or fluctuating for 3 to 6 weeks, example
Nutrient medium - Assuming that a shoot apex is cultured, then basal medium + a low level of cytokinin to promote shoot elongation and axillary bud development, gibberellin may also favor shoot elongation
Thermotherapy and Tissue Culture Procedures for Obtaining Disease-free Stock Plants
II. Factors affecting recovery of disease-free plants
Treatment of the donor plant - treatments that favor differential growth of the plant over the disease organism
Gibberellin or etiolation treatments – facilitate more rapid growth of the shoot
Thermotherapy treatment of plants – reduces pathogen growth (viral replication), 35 to 42 C constant or fluctuating for 3 to 6 weeks
Nutrient medium – shoot apex culture
basal medium + a low level of cytokinin to promote shoot elongation and axillary bud development, gibberellin may also favor shoot elongation, example
shoot apical meristems require more complex media
Asparagus Shoot Apex Development Stimulated by Low Cytokinin + Auxin
3. Root Cultures
I. Definition and Background
II. Explant, Media, Growth Conditions, and Reculture
III. Hairy Root Cultures
3. Root Cultures
I. Definition and Background
Roots growing autonomously in vitro
P R White established the first root culture (tomato) in 1933, culture is still maintained (1980), even though the primary root meristem has a determinate growth pattern
Principal use was to study the physiology and metabolism of roots, and primary root determinate growth patterns
Transformation to produce hairy root cultures has refocused interest on root secondary product biosynthesis
II. Explant, Media, Growth Conditions, and Reculture
Explant – primary root of aseptic seedling, example
Media – basal (essential micro- and macronutrients, carbon source), thiamine, typically growth regulator autotrophic
Growth Conditions – liquid or semisolid medium, aeration is important
Reculture – terminal meristem has a finite (determinant) growth, culture is maintained by re-culturing lateral root segments
Root Culture Initiation
Seedling after germination in vitro, primary root without secondary roots
Excise the terminal 10 mm and culture into medium
II. Explant, Media, Growth Conditions, and Reculture
Explant – primary root of aseptic seedling
Media – basal (essential micro- and macronutrients, carbon source), thiamine, typically growth regulator autotrophic
Growth Conditions – liquid or semisolid medium, aeration is important
Reculture – terminal meristem has a finite (determinant) growth, culture is maintained by re-culturing lateral root segments, example
Root Culture Growth and Reculture
Tomato root cultures
Reculture by excising lateral root and inoculate into fresh medium
Reculture of a Root
III. Hairy Root Cultures
Hairy root cultures are capable of complete autonomous growth/proliferation because of Agrobacterium rhizogenes transformation including production of numerous lateral roots, example
Hairy root culture scale-up
Hairy Root Culture
III. Hairy Root Cultures
Hairy root cultures are capable of complete autonomous growth/proliferation because of Agrobacterium rhizogenes transformation including production of numerous lateral roots
Hairy root culture scale-up - The vigorous growth of these cultures has made scale-up by engineers feasible
Illustrated is the growth of hairy root culture, culture vessels for scale-up and types of products that have been produced by hairy root cultures, examples
Hairy Root Culture Fermentation Systems
Table 1.1. Examples of secondary metabolites produced by hairy roots.
Genus Metabolite Reference
Ajuga Hydroxyecydsone Tanaka and Matsumoto (1993)
Ambrosia Thiophenes Flores et al. (1988)
Armoracia Fusicoccin Babakov et al. (1995)
Artemisia Artemisinin Qin et al. (1994), Weathers et al. (1994), Jaziri et al. (1995)
Astragalus Astragalosides Hirotani et al. (1994)
Atropa Tropane alkaloids Kamada et al. (1986), Jung and Tepfer (1987), Sharp and Doran (1990)
Beta Betalain pigments Hamill et al. (1986), Taya et al. (1992, 1994)
Bidens Polyacetylenes Marchant (1988)
Brugmansia Tropane alkaloid Giulietti et al. (1993)
Campanula Polyacetylenes Tada et al. (1996)
Carthamus Thiophenes Flores et al. (1988)
Cassia AnthraquinonesPolyketide pigments
Asamizu et al. (1988)Ko et al. (1995)
Catharanthus Indole alkaloids Parr et al. (1988), Toivonen et al. (1989), Bhadra et al. (1993), Sim et al. (1994), Jung et al. (1994)
Centranthus Valepotriates Gränicher et al. (1995b)
Chaenactis Polyines Constabel and Towers (1988)
Cinchona Indole alkaloids Hamill et al. (1989)
Coreopsis Polyacetylenes Marchant (1988)
Datura Tropane alkaloids Payne et al. (1987), Christen et al. (1989), Robins et al. (1990), Parr et al. (1990), Dupraz et al. (1994), Rhodes et al. (1994)
Sesquiterpenes Furze et al. (1991)
Daucus FlavonoidsAnthocyanin
Bel-Rhlid et al. (1993)Kim et al. (1994)
Digitalis Cardioactive glycosides Saito et al. (1990)
Duboisia Tropane alkaloid Deno et al. (1987b), Mano et al. (1989), Yukimune et al. (1994)
Echinacea Alkamides Trypsteen et al. (1991)
Fragaria Polyphenol Motomori et al. (1995)
Glycyrrhiza Glycyrrhizin Ko et al. (1989)
Gynostemma
Saponin Fei et al. (1993)
Hyoscyamus Tropane alkaloids Flores and Filner (1985), Parr et al. (1990), Doerk-Schmitz et al. (1994)
Piperidone alkaloids Sesquiterpenes
Sauerwein et al. (1991)Signs and Flores (1989)
Lactuca Sesquiterpene lactones Kisiel et al. (1995), Song et al. (1996)
Leontopodium
Anthocyanins and essential oils
Hook (1994)
Linum Lignans Berlin et al. (1988)
Lippia Sesquiterpenes Sauerwein et al. (1991)
Lithospermum
Naphthoquinone (shikonin)
Shimomura et al. (1991), Sim and Chang (1993)
Lobelia Piperidine alkaloidPolyacetylenes
Yonemitsu et al. (1990)Jshimaru et al. (1994), Tada et al. (1995a), Yamanaka et al. (1996)
Lotus Condensed tannins Carron et al. (1994)
Nicotiana Pyridine alkaloids Sesquiterpenoids
Hamill et al. (1986), Parr and Hamill (1987), Hamill et al.(1990), Green et al. (1992), Larsen et al. (1993)Wibberley et al. (1994)
Panax Saponins Yoshikawa and Furuya (1987), Inomata et al. (1993)
Platycodon Polyacetylenes Tada et al. (1995b)
Podophyllum
Lignans Berlin et al. (1988)
Rauwolfia Indole alkaloids Benjamin et al, (1994)
Rubia Anthraquinone Sato et al (1991), van der Heijden et al. (1994), Kino-oka et al. (1994)
Rudbeckia Thiophenes Flores et al. (1988), Daimon and Mu (1995)
Salvia Diterpenoid Hu and Alfermann (1993)
Scoparia Methoxybenzoxazolinone Hayashi et al. (1994)
Scopolia Tropane alkaloids Mano et al. (1986), Parr et al (1990), Ahn et al. (1993)
Senecio* Pyrrolizidine Toppel et al. (1987), Hartmann and Toppel (1987)
Serratula Ecdysteroid Delbecque et al. (1995)
Sesamum Naphthoquinone Ogasawara et al. (1993)
Solanum Steroids Subroto and Doran (1994), Alvarez et al. (1994), Drewes and van Staden (1995b), Ikenaga et al. (1995), Yu et al. (1996)
Swainsona Swainsonine Ermayanti et al. (1994)
Tagetes Thiophenes Westcott (1988), Croes et al. (1989), Buitelaar et al. (1993), Talou et al. (1994), Jacobs et al. (1995)
Trichosanthes
Bryonolic acid Takeda et al. (1994)
Valeriana Valepotriates Iridoid diester
Gränicher et al. (1994)Gränicher et al. (1995a)
Withania Withanolides Banerjee et al. (1994)*In this case, fast growing root cultures were established in medium devoid of phytohormones without being transformed with A. rhizogenes. This serves to remind us that it is the fact that fully differentiated roots are being cultured, and not transformation by Ri T-DNA per se, which accounts for the large number of reports of secondary metabolite formation by hairy roots as indicated in Table 1.1.